MODULATION OF APOLIPOPROTEIN (a) EXPRESSION

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of apolipoprotein(a). The compositions comprise oligonucleotides, targeted to nucleic acid encoding apolipoprotein(a). Methods of using these compounds for modulation of apolipoprotein(a) expression and for diagnosis and treatment of disease associated with expression of apolipoprotein(a) are provided.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/726,286, filed Mar. 17, 2010, is a Continuation of U.S. patentapplication Ser. No. 10/559,647, filed Jul. 31, 2006, which is a USNational Phase Application under 35 USC 371 of PCT/US2004/014540 filedon Jun. 2, 2004, which claims priority to U.S. provisional patentapplication No. 60/475,402, filed Jun. 2, 2003 and U.S. patentapplication Ser. No. 10/684,440, filed Oct. 15, 2003, each of which isincorporated by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledISPH0595USC5_ST25.txt, created on Jan. 21, 2014 which is 168 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of apolipoprotein(a).

Lipoproteins are globular, micelle-like particles that consist of anon-polar core of acylglycerols and cholesteryl esters, surrounded by anamphiphilic coating consisting of protein, phospholipid and cholesterol.Lipoproteins have been classified into five broad categories on thebasis of their functional and physical properties: chylomicrons (whichtransport dietary lipids from intestine to tissues), very low densitylipoproteins (VLDL), intermediate density lipoproteins (IDL). Lowdensity lipoproteins (LDL), (all of which transport triacylglycerols andcholesterol from the liver to tissues), and high density lipoproteins(HDL) (which transport endogenous cholesterol from tissues to theliver). Lipoprotein particles undergo continuous metabolic processingand have variable properties and compositions. Lipoprotein densitiesincrease without decreasing particle diameter because the density oftheir outer coatings is less than that of the inner core. The proteincomponents of lipoproteins are known as apolipoproteins. At least nineapolipoproteins are distributed in significant amounts among the varioushuman lipoproteins.

Lipoprotein (a) (also known as Lp(a)) is a cholesterol rich particle ofthe proatherogenic LDL class. Since Lp(a) is found only in Old Worldprimates and European hedgehogs, it has been suggested that it

does not play an essential role in lipid and lipoprotein metabolism.Most studies have shown that high concentrations of Lp(a) are stronglyassociated with increased risk of cardiovascular disease (Rainwater andKammerer, J. Exp. Zool., 1998, 282, 54-61). These observations havestimulated numerous studies in humans and other primates to investigatethe factors that control Lp(a) concentrations and physiologicalproperties (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61).

Lp(a) contains two disulfide-linked distinct proteins, apolipoprotein(a)(or ApoA) and apolipoprotein B (or ApoB) (Rainwater and Kammerer, J.Exp. Zool., 1998, 282, 54-61). Apolipoprotein(a) is a uniqueapolipoprotein encoded by the LPA gene which has been shown toexclusively control the physiological concentrations of Lp(a) (Rainwaterand Kammerer, J. Exp. Zool., 1998, 282, 54-61). It varies in size due tointerallelic differences in the number of tandemly repeatedKringle-4-encoding 5.5 kb sequences in the LPA gene (Rainwater andKammerer, J. Exp. Zool., 1998, 282, 54-61).

Cloning of human apolipoprotein(a) in 1987 revealed homology to humanplasminogen (McLean et al., Nature, 1987, 330, 132-137). The gene locusLPA encoding apolipoprotein(a) was localized to chromosome 6q26-27, inclose proximity to the homologous gene for plasminogen (Frank et al.,Hum. Genet., 1988, 79, 352-356).

Transgenic mice expressing human apolipoprotein(a) were found to be moresusceptible than control mice to the development of lipid-staininglesions in the aorta. Consequently, apolipoprotein(a) is co-localizedwith lipid deposition in the artery walls (Lawn et al., Nature, 1992,360, 670-672). As an extension of these studies, it was established thatthe major in vivo action of apolipoprotein(a) is inhibition of theconversion of plasminogen to plasmin which causes decreased activationof latent transforming growth factor-beta. Since transforming growthfactor-beta is a negative regulator of smooth muscle cell migration andproliferation, inhibition of plasminogen activation indicates a possiblemechanism for apolipoprotein(a) induction of atherosclerotic lesions(Grainger et al., Nature, 1994, 370, 460-462).

Elevated plasma levels of Lp(a), caused by increased expression ofapolipoprotein(a), are associated with increased risk foratherosclerosis and its manifestations, which includehypercholesterolemia (Seed et al., N. Engl. J. Med., 1990, 322,1494-1499), myocardial infarction (Sandkamp et al., Clin. Chem., 1990,36, 20-23), and thrombosis (Nowak-Gottl et al., Pediatrics, 1997, 99,E11).

Moreover, the plasma concentration of Lp(a) is strongly influenced byheritable factors and is refractory to most drug and dietarymanipulation (Katan and Beynen, Am. J. Epidemiol., 1987, 125, 387-399;Vessby et al., Atherosclerosis, 1982, 44, 61-71.). Pharmacologic therapyof elevated Lp(a) levels has been only moderately successful andapheresis remains the most effective therapeutic modality (Hajjar andNachman, Annu. Rev. Med., 1996, 47, 423-442).

Morishita et al. reported the use of three ribozyme oligonucleotidesagainst apolipoprotein(a) for inhibition of apolipoprotein(a) expressionin HepG2 cells (Morishita et al., Circulation, 1998, 98, 1898-1904).

U.S. Pat. No. 5,721,138 refers to nucleotide sequences encoding thehuman apolipoprotein(a) gene 5′-regulatory region and isolatednucleotide sequences comprising at least thirty consecutivecomplementary nucleotides from human apolipoprotein(a) from nucleotidepositions 208 to 1448 (Lawn, 1998).

To date, investigative and therapeutic strategies aimed at inhibitingapolipoprotein(a) function have involved the previously cited use ofLp(a) apheresis and ribozyme oligonucleotides. No existing drugs areavailable to specifically lower lipoprotein(a) levels in humans, andonly limited models exist in which to perform drug discovery.Consequently, there remains a long-felt need for additional agents andmethods capable of effectively modulating, e.g., inhibiting,apolipoprotein(a) function, and particularly a need for agents capableof safe and efficacious administration to lower alipoprotein(a) levelsin patients at risk for the development of coronary artery disease.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of apolipoprotein(a). Such novel compositions and methodsenable research into the pathways of plasminogen and apolipoprotein(a),as well as other lipid metabolic processes. Such novel compositions andmethods are useful in assessing the toxicity of chemical andpharmaceutical compounds on apolipoprotein(a) function, plasminogen orother lipid metabolic processes. Such novel compositions and methods areuseful for drug discovery and for the treatment of cardiovascularconditions, including myocardial infarction and atherosclerosis, amongothers.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products, and is uniquely useful in a numberof therapeutic, diagnostic, and research applications for the modulationof apolipoprotein(a) expression.

In particular, this invention relates to compounds, particularlyoligonucleotide compounds, which, in preferred embodiments, hybridizewith nucleic acid molecules or sequences encoding apolipoprotein(a).Such compounds are shown herein to modulate the expression ofapolipoprotein(a). Additionally disclosed are embodiments ofoligonucleotide compounds that hybridize with nucleic acid moleculesencoding apolipoprotein(a) in preference to nucleic acid molecules orsequences encoding plasminogen.

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding apolipoprotein(a), and which modulate the expression ofapolipoprotein(a). Pharmaceutical and other compositions comprising thecompounds of the invention are also provided.

Further provided are methods of screening for modulators ofapolipoprotein(a) and methods of modulating the expression ofapolipoprotein(a) in cells, tissues or animals comprising contactingsaid cells, tissues or animals with one or more of the compounds orcompositions of the invention. In these methods, the cells or tissuesmay be contacted in vivo. Alternatively, the cells or tissues may becontacted ex vivo.

Methods of treating an animal, particularly a human, having, suspectedof having, or being prone to a disease or condition associated withexpression of apolipoprotein(a) are also set forth herein. Such methodscomprise administering a therapeutically or prophylactically effectiveamount of one or more of the compounds or compositions of the inventionto the person in need of treatment.

In one aspect, the invention provides the use of a compound orcomposition of the invention in the manufacture of a medicament for thetreatment of any and all conditions disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species, for use in modulating the function or effect of nucleicacid molecules encoding apolipoprotein(a). This is accomplished byproviding oligonucleotides that specifically hybridize with one or morenucleic acid molecules encoding apolipoprotein(a). As used herein, theterms “target nucleic acid” and “nucleic acid molecule encodingapolipoprotein(a)” have been used for convenience to encompass DNAencoding apolipoprotein(a), RNA (including pre-mRNA and mRNA or portionsthereof) transcribed from such DNA, and also cDNA derived from such RNA.The hybridization of a compound of this invention with its targetnucleic acid is generally referred to as “antisense”. Antisensetechnology is emerging as an effective means of reducing the expressionof specific gene products and is uniquely useful in a number oftherapeutic, diagnostic and research applications involving modulationof apolipoprotein(a) expression.

Consequently, the preferred mechanism believed to be included in thepractice of some preferred embodiments of the invention is referred toherein as “antisense inhibition.” Such antisense inhibition is typicallybased upon hydrogen bonding-based hybridization of oligonucleotidestrands or segments, such that at least one strand or segment iscleaved, degraded, or otherwise rendered inoperable. In this regard, itis presently preferred to target specific nucleic acid molecules andtheir functions for such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA, which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofapolipoprotein(a). In the context of the present invention, “modulation”and “modulation of expression” mean either an increase (stimulation) ora decrease (inhibition) in the amount or levels of a nucleic acidmolecule encoding the gene, e.g., DNA or RNA. Inhibition is often thepreferred form of modulation of expression and mRNA is often a preferredtarget nucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases that pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired. Such conditionsinclude, e.g., physiological conditions in the case of in vivo assays ortherapeutic treatment, and conditions in which assays are performed inthe case of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances. In the context of thisinvention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound) is capable of hydrogen bonding with a nucleobase ata certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases that can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

The sequence of an antisense compound can be, but need not necessarilybe, 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event. In one embodiment of thisinvention, the antisense compounds of the present invention comprise atleast 70%, or at least 75%, or at least 80%, or at least 85% sequencecomplementarity to a target region within the target nucleic acid. Inother embodiments, the antisense compounds of the present inventioncomprise at least 90% sequence complementarity and even comprise atleast 95% or at least 99% sequence complementarity to the target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases, and need not be contiguous to each other or tocomplementary nucleobases. As such, an antisense compound which is 18nucleobases in length having 4 (four) noncomplementary nucleobases whichare flanked by two regions of complete complementarity with the targetnucleic acid would have 77.8% overall complementarity with the targetnucleic acid and would thus fall within the scope of the presentinvention. Percent complementarity of an antisense compound with aregion of a target nucleic acid can be determined routinely using BLASTprograms (basic local alignment search tools) and PowerBLAST programsknown in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410;Zhang and Madden, Genome Res., 1997, 7, 649-656).

Percent homology, sequence identity, or complementarity can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Insome embodiments, homology, sequence identity, or complementaritybetween the oligomeric and target is between about 50% to about 60%. Insome embodiments, homology, sequence identity, or complementarity isbetween about 60% to about 70%. In other embodiments, homology, sequenceidentity, or complementarity is between about 70% and about 80%. Instill other embodiments, homology, sequence identity, or complementarityis between about 80% and about 90%. In yet other embodiments, homology,sequence identity, or complementarity is about 90%, about 92%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100%.

B. Compounds of the Invention

According to the present invention, “compounds” include antisenseoligomeric compounds, antisense oligonucleotides, siRNAs, external guidesequence (EGS) oligonucleotides, alternate splicers, and otheroligomeric compounds that hybridize to at least a portion of the targetnucleic acid. As such, these compounds may be introduced in the form ofsingle-stranded, double-stranded, partially single-stranded, or circularoligomeric compounds. Specifically excluded from the definition of“compounds” herein are ribozymes that contain internal or external“bulges” that do not hybridize to the target sequence. Once introducedto a system, the compounds of the invention may elicit the action of oneor more enzymes or structural proteins to effect modification of thetarget nucleic acid.

One non-limiting example of such an enzyme is RNase H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds that are“DNA-like” elicit RNase H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While one form of antisense compound is a single-stranded antisenseoligonucleotide, in many species the introduction of double-strandedstructures, such as double-stranded RNA (dsRNA) molecules, has beenshown to induce potent and specific antisense-mediated reduction of thefunction of a gene or its associated gene products. This phenomenonoccurs in both plants and animals and is believed to have anevolutionary connection to viral defense and transposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell, 1995, 81, 611-620). The primary interference effects ofdsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci.USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, the single-stranded RNAoligomers of antisense polarity of the dsRNAs have been reported to bethe potent inducers of RNAi (Tijsterman et al., Science, 2002, 295,694-697).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA), or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars, and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid, and increased stability in the presence of nucleases.

The oligonucleotides of the present invention also include modifiedoligonucleotides in which a different base is present at one or more ofthe nucleotide positions in the oligonucleotide. For example, if thefirst nucleotide is an adenosine, modified oligonucleotides may beproduced that contain thymidine, guanosine or cytidine at this position.This may be done at any of the positions of the oligonucleotide. Theseoligonucleotides are then tested using the methods described herein todetermine their ability to inhibit expression of apolipoprotein(a) mRNA.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to, oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention comprise from about 8 toabout 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).One of ordinary skill in the art will appreciate that the inventionembodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, or 80 nucleobases in length.

In one embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another embodiment, the compounds of the invention are 15 to 30nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

In another embodiment, compounds of this invention are oligonucleotidesfrom about 12 to about 50 nucleobases. In another embodiment, compoundsof this invention comprise from about 15 to about 30 nucleobases.

In another embodiment, the antisense compounds comprise at least 8contiguous nucleobases of an antisense compound disclosed herein.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary compounds include oligonucleotide sequences that comprise atleast the 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred antisense compounds (the remaining nucleobasesbeing a consecutive stretch of the same oligonucleotide beginningimmediately upstream of the 5′-terminus of the antisense compound thatis specifically hybridizable to the target nucleic acid, and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases).Similarly, exemplary antisense compounds are represented byoligonucleotide sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredantisense compounds (the remaining nucleobases being a consecutivestretch of the same oligonucleotide beginning immediately downstream ofthe 3′-terminus of the antisense compound that is specificallyhybridizable to the target nucleic acid and continuing until theoligonucleotide contains about 8 to about 80 nucleobases).

Exemplary compounds of this invention may be found identified in theExamples and listed in Tables 1 and 7. In addition to oligonucleotidecompounds that bind to target sequences of apolipoprotein(a) in general,there are also exemplified oligonucleotide compounds of this inventionthat bind to target nucleotide sequences of apolipoprotein(a), but donot bind to, or do not bind preferentially to, sequences of plasminogendue to lack of homology between the two nucleic acid molecules or asufficient number of mismatches in the target sequences. These lattercompounds are also useful in various therapeutic methods of thisinvention. Examples of antisense compounds to such ‘mismatched’ targetsequences as described above include SEQ ID NO: 12 and SEQ ID NO: 23 ofTable 1 below. See, also, the discussion of target regions below.

One having skill in the art armed with the exemplary antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further useful antisense compounds.

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes apolipoprotein(a).

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes having translation initiation codons withthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG; and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA transcribed from a geneencoding apolipoprotein(a), regardless of the sequence(s) of suchcodons. A translation termination codon (or “stop codon”) of a gene mayhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions that may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Another target region includes the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene). Still another target regionincludes the 3′ untranslated region (3′UTR), known in the art to referto the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA (or correspondingnucleotides on the gene). The 5′ cap site of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap site. Another target region for thisinvention is the 5′ cap region.

Accordingly, the present invention provides antisense compounds thattarget a portion of nucleotides 1-2480 as set forth in SEQ ID NO: 4. Inanother embodiment, the antisense compounds target at least an8-nucleobase portion of nucleotides 1-45, comprising the 5′UTR as setforth in SEQ ID NO: 4. In another embodiment, the antisense compoundstarget at least an 8-nucleobase portion of nucleotides 13593-13938,comprising the 3′UTR as set forth in SEQ ID NO: 4. In anotherembodiment, the antisense compounds target at least an 8-nucleobaseportion of nucleotides 46-13592, comprising the coding region as setforth in SEQ ID NO: 4. In still other embodiments, the antisensecompounds target at least an 8-nucleobase portion of a “preferred targetsegment” (as defined herein) as set forth in Table 2.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence, resulting in exon-exon junctions at thesites where exons are joined. Targeting exon-exon junctions can beuseful in situations where the overproduction of a normal splice productis implicated in disease, or where the overproduction of an aberrantsplice product is implicated in disease. In one embodiment, targetingsplice sites, i.e., intron-exon junctions or exon-intron junctions, isparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular splice product isimplicated in disease. An aberrant fusion junction due to rearrangementor deletion is another embodiment of a target site. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources known as “fusion transcripts” are also suitabletarget sites. Introns can be effectively targeted using antisensecompounds targeted to, for example, DNA or pre-mRNA.

Alternative RNA transcripts can be produced from the same genomic regionof DNA. These alternative transcripts are generally known as “variants”.More specifically, “pre-mRNA variants” are transcripts produced from thesame genomic DNA that differ from other transcripts produced from thesame genomic DNA in either their start or stop position and contain bothintronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants, and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

Variants can be produced through the use of alternative signals to startor stop transcription. Pre-mRNAs and mRNAs can possess more that onestart codon or stop codon. Variants that originate from a pre-mRNA ormRNA that use alternative start codons are known as “alternative startvariants” of that pre-mRNA or mRNA. Those transcripts that use analternative stop codon are known as “alternative stop variants” of thatpre-mRNA or mRNA. One specific type of alternative stop variant is the“polyA variant” in which the multiple transcripts produced result fromthe alternative selection of one of the “polyA stop signals” by thetranscription machinery, thereby producing transcripts that terminate atunique polyA sites. Within the context of the invention, the types ofvariants described herein are also embodiments of target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid that are accessible forhybridization.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the target segmentsillustrated herein will be able, without undue experimentation, toidentify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In various embodiments of this invention, the oligomeric compounds aretargeted to regions of a target apolipoprotein(a) nucleobase sequence,such as those disclosed herein. All regions of the target nucleobasesequence to which an oligomeric antisense compound can be targeted,wherein the regions are greater than or equal to 8 and less than orequal to 80 nucleobases, are described as follows:

Let R(m, n+m−1) be a region from a target nucleobase sequence, where “n”is the 5′-most nucleobase position of the region, where “n+m−1” is the3′-most nucleobase position of the region and where “m” is the length ofthe region. A set “S(m)”, of regions of length “m” is defined as theregions where n ranges from 1 to L−m+1, where L is the length of thetarget nucleobase sequence and L>m. A set, “A”, of all regions can beconstructed as a union of the sets of regions for each length from wherem is greater than or equal to 8 and is less than or equal to 80.

This set of regions can be represented using the following mathematicalnotation:

$A = {\bigcup\limits_{m}{S(m)}}$ where  m ∈ N|8 ≤ m ≤ 80 andS(m) = {R_(n, n + m − 1)|n ∈ {1, 2, 3, …  , L − m + 1}}

where the mathematical operator | indicates “such that”,

where the mathematical operator ε indicates “a member of a set” (e.g.yεZ indicates that element y is a member of set Z),

where x is a variable,

where N indicates all natural numbers, defined as positive integers,

and where the mathematical operator ∪ indicates “the union of sets”.

For example, the set of regions for m equal to 8, 9 and 80 can beconstructed in the following manner. The set of regions, each 8nucleobases in length, S(m=8), in a target nucleobase sequence 100nucleobases in length (L=100), beginning at position 1 (n=1) of thetarget nucleobase sequence, can be created using the followingexpression:

S(8)={R _(1,8) |nε{1,2,3, . . . ,93}}

and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10,4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21,15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31,25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41,35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51,45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61,55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71,65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81,75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91,85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:

S(20)={R _(1,20) |nε{1,2,3, . . . ,81}}

and describes the set of regions comprising nucleobases 1-20, 2-21,3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32,14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42,24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52,34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62,44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72,54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82,64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92,74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:

S(80)={R _(1,80) |nε{1,2,3, . . . ,21}}

and describes the set of regions comprising nucleobases 1-80, 2-81,3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92,14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

Thus, in this example, A would include regions 1-8, 2-9, 3-10-93-100,1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The union of these aforementioned example sets and other sets forlengths from 10 to 19 and 21 to 79 can be described using themathematical expression:

$A = {\bigcup\limits_{m}{S(m)}}$

where ∪ represents the union of the sets obtained by combining allmembers of all sets.

The mathematical expressions described herein define all possible targetregions in a target nucleobase sequence of any length L, where theregion is of length m, and where m is greater than or equal to 8 andless than or equal to 80 nucleobases, and where m is less than L, andwhere n is less than L−m+1.

In one embodiment, the oligonucleotide compounds of this invention are100% complementary to these sequences or to small sequences found withineach of the above listed sequences. In another embodiment theoligonucleotide compounds have from at least 3 or 5 mismatches per 20consecutive nucleobases in individual nucleobase positions to thesetarget regions. Still other compounds of the invention are targeted tooverlapping regions of the above-identified portions of theapolipoprotein(a) sequence.

In still another embodiment, target regions include those portions ofthe apolipoprotion(a) sequence that do not overlap with plasminogensequences. For example, among such apolipoprotein(a) target sequencesare included those found within the following nucleobase sequences:10624-10702, 10963-11036, 11325-11354, 11615-11716, 11985-12038,12319-12379, 13487-13491, and 13833-13871. As a further example, targetsequences of apolipoprotein(a) that have at least 6 mismatches with thesequence of plasminogen over at least 20 consecutive nucleotides aredesirable targets for antisense compounds that bind preferentially toapolipoprotein(a) rather than to plasminogen. Such target sequences canreadily be identified by a BLAST comparison of the two GENBANK®sequences of plasminogen (e.g., GENBANK® Accession No. NM_(—)000301) andapolipoprotein(a) (e.g., GENBANK® Accession No. NM_(—)005577.1).

In still another embodiment, the target regions include portions of theapolipoprotein (a) sequence that overlap with portions of theplasminogen or apolipoprotein B sequence, but to which antisensecompounds bind to inhibit apolipoprotein (a) but do not inhibit, to anyappreciable degree, plasminogen and/or apolipoprotein B. Such targetsmay be obtained from the target regions of SEQ ID NOs: 46, 54, 56, 57,59, 60, 61, 62, 64, 67, 68 and 69 of Table 2. These target regions arebound by antisense oligonucleotides of SEQ ID Nos: 11, 23, 28, 30, 31,33, 34, 35, 36, 39, 42, 43, and 45, for example, which inhibitapolipoprotein(a) but not a second protein, which is plasminogen (seeExample 22) or apolipoprotein B (see Example 23).

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of apolipoprotein(a). “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding apolipoprotein(a) and which comprise at least an8-nucleobase portion that is complementary to a preferred targetsegment. The screening method comprises the steps of contacting apreferred target segment of a nucleic acid molecule encodingapolipoprotein(a) with one or more candidate modulators, and selectingfor one or more candidate modulators which decrease or increase theexpression of a nucleic acid molecule encoding apolipoprotein(a). Onceit is shown that the candidate modulator or modulators are capable ofmodulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding apolipoprotein(a), the modulator may thenbe employed in further investigative studies of the function ofapolipoprotein(a), or for use as a research, diagnostic, or therapeuticagent in accordance with the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocesssing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature,1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons etal., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282,430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir etal., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15,188-200). For example, such double-stranded moieties have been shown toinhibit the target by the classical hybridization of the antisensestrand of the duplex to the target, thereby triggering enzymaticdegradation of the target (Tijsterman et al., Science, 2002, 295,694-697).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between apolipoprotein(a) and a disease state, phenotype, orcondition. These methods include detecting or modulatingapolipoprotein(a) comprising contacting a sample, tissue, cell, ororganism with the compounds of the present invention, measuring thenucleic acid or protein level of apolipoprotein(a) and/or a relatedphenotypic or chemical endpoint at some time after treatment, andoptionally comparing the measured value to a non-treated sample orsample treated with a further compound of the invention. These methodscan also be performed in parallel or in combination with otherexperiments to determine the function of unknown genes for the processof target validation or to determine the validity of a particular geneproduct as a target for treatment or prevention of a particular disease,condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention are utilized for diagnostics,therapeutics, and prophylaxis, and as research reagents and componentsof kits. Furthermore, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes orto distinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics and in various biological systems, thecompounds of the present invention, either alone or in combination withother compounds or therapeutics, are used as tools in differentialand/or combinatorial analyses to elucidate expression patterns of aportion or the entire complement of genes expressed within cells andtissues.

As used herein the term “biological system” or “system” is defined asany organism, cell, cell culture or tissue that expresses, or is madecompetent to express products of the LPA gene. These include, but arenot limited to, humans, transgenic animals, cells, cell cultures,tissues, xenografts, transplants and combinations thereof.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundsthat affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000 480, 17-24;Celis, et al., FEBS Lett., 2000 480, 2-16), SAGE (serial analysis ofgene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425),READS (restriction enzyme amplification of digested cDNAs) (Prashar andWeissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total geneexpression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A.,2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBSLett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20,2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBSLett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encodingapolipoprotein(a). Primers and probes disclosed herein are useful inmethods requiring the specific detection of nucleic acid moleculesencoding apolipoprotein(a) and in the amplification of said nucleic acidmolecules for detection or for use in further studies ofapolipoprotein(a). Hybridization of the primers and probes with anucleic acid encoding apolipoprotein(a) can be detected by means knownin the art. Such means may include conjugation of an enzyme to theprimers and probes, radiolabelling of the primers and probes, or anyother suitable detection means. Kits using such detection means fordetecting the level of apolipoprotein(a) in a sample may also beprepared.

The invention further provides for the use of a compound or compositionof the invention in the manufacture of a medicament for the treatment ofany and all conditions disclosed herein.

The specificity and sensitivity of antisense are also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs have beensafely and effectively administered to humans and numerous clinicaltrials are underway. It is thus established that antisense compounds canbe useful therapeutic modalities that can be configured to be useful intreatment regimes for the treatment of cells, tissues and animals,especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofapolipoprotein(a) is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of aapolipoprotein(a) inhibitor. The apolipoprotein(a) inhibitors of thepresent invention effectively inhibit the activity of theapolipoprotein(a) protein or inhibit the expression of theapolipoprotein(a) protein. In one embodiment, the activity or expressionof apolipoprotein(a) in an animal is inhibited by about 10%. Preferably,the activity or expression of apolipoprotein(a) in an animal isinhibited by about 30%. More preferably, the activity or expression ofapolipoprotein(a) in an animal is inhibited by 50% or more. Thus, theoligomeric compounds modulate expression of apolipoprotein(a) mRNA by atleast 10%, by at least 20%, by at least 25%, by at least 30%, by atleast 40%, by at least 50%, by at least 60%, by at least 70%, by atleast 75%, by at least 80%, by at least 85%, by at least 90%, by atleast 95%, by at least 98%, by at least 99%, or by 100%.

For example, the reduction of the expression of apolipoprotein(a) may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. Preferably, the cells contained within saidfluids, tissues or organs being analyzed contain a nucleic acid moleculeencoding apolipoprotein(a) protein and/or the apolipoprotein(a) proteinitself. For example, apolipoprotein(a) is produced in the liver, and canbe found in normal and atherosclerotic vessel walls.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages—Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone) of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Further embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(m)O]CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-β-methoxyethyl (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-methoxyethoxy or 2′-MOE) (Martin etal., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Afurther preferred modification includes 2′-dimethylaminooxyethoxy, i.e.,a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-O—CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920; certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further modification of the sugar includes Locked Nucleic Acids (LNAs)in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom ofthe sugar ring, thereby forming a bicyclic sugar moiety. The linkage ispreferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in International Patent Publication Nos. WO 98/39352 and WO99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deazaadenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941; certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, the entire disclosures of which areincorporated herein by reference. Conjugate moieties include, but arenot limited to, lipid moieties such as a cholesterol moiety, cholicacid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, analiphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999), which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941; certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of oligomeric compounds toenhance properties such as for example nuclease stability. Included instabilizing groups are cap structures. By “cap structure or terminal capmoiety” is meant chemical modifications, which have been incorporated ateither terminus of oligonucleotides (see for example Wincott et al.,International Patent Publication No. WO 97/26270, incorporated byreference herein). These terminal modifications protect the oligomericcompounds having terminal nucleic acid molecules from exonucleasedegradation, and can help in delivery and/or localization within a cell.The cap can be present at the 5′-terminus (5′-cap) or at the 3′-terminus(3′-cap) or at both termini. In non-limiting examples, the 5′-capincludes inverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International Patent Publication No. WO 97/26270,incorporated by reference herein).

Particularly preferred 3′-cap structures of the present inventioninclude, for example 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate; bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in International Patent Publication No. WO 03/004602,published Jan. 16, 2003.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds that arechimeric compounds. “Chimeric” antisense compounds, or “chimeras,” inthe context of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases such as RNaseL, which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Preferred chimeric oligonucleotides are those disclosed in the Examplesherein. Particularly preferred chimeric oligonucleotides are thosereferred to as ISIS 144367, ISIS 144368, ISIS 144379, ISIS 144381, andISIS 144396.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Chimeric antisense compounds can be of several different types. Theseinclude a first type wherein the “gap” segment of linked nucleosides ispositioned between 5′ and 3′ “wing” segments of linked nucleosides and asecond “open end” type wherein the “gap” segment is located at eitherthe 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotidesof the first type are also known in the art as “gapmers” or gappedoligonucleotides. Oligonucleotides of the second type are also known inthe art as “hemimers” or “wingmers”.

Such compounds have also been referred to in the art as hybrids. In agapmer that is 20 nucleotides in length, a gap or wing can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides inlength. In one embodiment, a 20-nucleotide gapmer is comprised of a gap8 nucleotides in length, flanked on both the 5′ and 3′ sides by wings 6nucleotides in length. In another embodiment, a 20-nucleotide gapmer iscomprised of a gap 10 nucleotides in length, flanked on both the 5′ and3′ sides by wings 5 nucleotides in length. In another embodiment, a20-nucleotide gapmer is comprised of a gap 12 nucleotides in lengthflanked on both the 5′ and 3′ sides by wings 4 nucleotides in length. Ina further embodiment, a 20-nucleotide gapmer is comprised of a gap 14nucleotides in length flanked on both the 5′ and 3′ sides by wings 3nucleotides in length. In another embodiment, a 20-nucleotide gapmer iscomprised of a gap 16 nucleotides in length flanked on both the 5′ and3′ sides by wings 2 nucleotides in length. In a further embodiment, a20-nucleotide gapmer is comprised of a gap 18 nucleotides in lengthflanked on both the 5′ and 3′ ends by wings 1 nucleotide in length.Alternatively, the wings are of different lengths, for example, a20-nucleotide gapmer may be comprised of a gap 10 nucleotides in length,flanked by a 6-nucleotide wing on one side (5′ or 3′) and a 4-nucleotidewing on the other side (5′ or 3′).

In a hemimer, an “open end” chimeric antisense compound, 20 nucleotidesin length, a gap segment, located at either the 5′ or 3′ terminus of theoligomeric compound, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18 or 19 nucleotides in length. For example, a20-nucleotide hemimer can have a gap segment of 10 nucleotides at the 5′end and a second segment of 10 nucleotides at the 3′ end. Alternatively,a 20-nucleotide hemimer can have a gap segment of 10 nucleotides at the3′ end and a second segment of 10 nucleotides at the 5′ end.

Representative United States patents that teach the preparation of suchhybrid structures include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922;certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756; each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes that are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids. When incorporated into liposomes, these specialized lipidsresult in liposomes with enhanced circulation lifetimes relative toliposomes lacking such specialized lipids. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome comprises one or more glycolipids or isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. Liposomes and their uses are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e., route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298, filed May 20,1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. Published Patent Application No. 2003/0040497 (Feb. 27,2003) and its parent applications; U.S. Published Patent Application No.2003/0027780 (Feb. 6, 2003) and its parent applications; and U.S. patentapplication Ser. No. 10/071,822, filed Feb. 8, 2002, each of which isincorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Oligonucleotides may be formulated for delivery in vivo in an acceptabledosage form, e.g. as parenteral or non-parenteral formulations.Parenteral formulations include intravenous (IV), subcutaneous (SC),intraperitoneal (IP), intravitreal and intramuscular (IM) formulations,as well as formulations for delivery via pulmonary inhalation,intranasal administration, topical administration, etc. Non-parenteralformulations include formulations for delivery via the alimentary canal,e.g. oral administration, rectal administration, intrajejunalinstillation, etc. Rectal administration includes administration as anenema or a suppository. Oral administration includes administration as acapsule, a gel capsule, a pill, an elixir, etc.

In some embodiments, an oligonucleotide may be administered to a subjectvia an oral route of administration. The subject may be an animal or ahuman (man). An animal subject may be a mammal, such as a mouse, rat,mouse, a rat, a dog, a guinea pig, a monkey, a non-human primate, a cator a pig. Non-human primates include monkeys and chimpanzees. A suitableanimal subject may be an experimental animal, such as a mouse, a rat, adog, a monkey, a non-human primate, a cat or a pig.

In some embodiments, the subject may be a human. In certain embodiments,the subject may be a human patient in need of therapeutic treatment asdiscussed in more detail herein. In certain embodiments, the subject maybe in need of modulation of expression of one or more genes as discussedin more detail herein. In some particular embodiments, the subject maybe in need of inhibition of expression of one or more genes as discussedin more detail herein. In particular embodiments, the subject may be inneed of modulation, i.e. inhibition or enhancement, of apolipoprotein(a)in order to obtain therapeutic indications discussed in more detailherein.

In some embodiments, non-parenteral (e.g. oral) oligonucleotideformulations according to the present invention result in enhancedbioavailability of the oligonucleotide. In this context, the term“bioavailability” refers to a measurement of that portion of anadministered drug which reaches the circulatory system (e.g. blood,especially blood plasma) when a particular mode of administration isused to deliver the drug. Enhanced bioavailability refers to aparticular mode of administration's ability to deliver oligonucleotideto the peripheral blood plasma of a subject relative to another mode ofadministration. For example, when a non-parenteral mode ofadministration (e.g. an oral mode) is used to introduce the drug into asubject, the bioavailability for that mode of administration may becompared to a different mode of administration, e.g. an IV mode ofadministration. In some embodiments, the area under a compound's bloodplasma concentration curve (AUC₀) after non-parenteral (e.g. oral,rectal, intrajejunal) administration may be divided by the area underthe drug's plasma concentration curve after intravenous (i.v.)administration (AUC_(iv)) to provide a dimensionless quotient (relativebioavailability, RB) that represents fraction of compound absorbed viathe non-parenteral route as compared to the IV route. A composition'sbioavailability is said to be enhanced in comparison to anothercomposition's bioavailability when the first composition's relativebioavailability (RB₁) is greater than the second composition's relativebioavailability (RB₂).

In general, bioavailability correlates with therapeutic efficacy when acompound's therapeutic efficacy is related to the blood concentrationachieved, even if the drug's ultimate site of action is intracellular(van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300).Bioavailability studies have been used to determine the degree ofintestinal absorption of a drug by measuring the change in peripheralblood levels of the drug after an oral dose (DiSanto, Chapter 76 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 1451-1458).

In general, an oral composition's bioavailability is said to be“enhanced” when its relative bioavailability is greater than thebioavailability of a composition substantially consisting of pureoligonucleotide, i.e. oligonucleotide in the absence of a penetrationenhancer.

Organ bioavailability refers to the concentration of compound in anorgan. Organ bioavailability may be measured in test subjects by anumber of means, such as by whole-body radiography. Organbioavailability may be modified, e.g. enhanced, by one or moremodifications to the oligonucleotide, by use of one or more carriercompounds or excipients, etc. as discussed in more detail herein. Ingeneral, an increase in bioavailability will result in an increase inorgan bioavailability.

Oral oligonucleotide compositions according to the present invention maycomprise one or more “mucosal penetration enhancers,” also known as“absorption enhancers” or simply as “penetration enhancers.”Accordingly, some embodiments of the invention comprise at least oneoligonucleotide in combination with at least one penetration enhancer.In general, a penetration enhancer is a substance that facilitates thetransport of a drug across mucous membrane(s) associated with thedesired mode of administration, e.g. intestinal epithelial membranes.Accordingly, it is desirable to select one or more penetration enhancersthat facilitate the uptake of an oligonucleotide, without interferingwith the activity of the oligonucleotide, and in such a manner theoligonucleotide may be introduced into the body of an animal withoutunacceptable side-effects such as toxicity, irritation or allergicresponse.

Embodiments of the present invention provide compositions comprising oneor more pharmaceutically acceptable penetration enhancers, and methodsof using such compositions, which result in the improved bioavailabilityof oligonucleotides administered via non-parenteral modes ofadministration. Heretofore, certain penetration enhancers have been usedto improve the bioavailability of certain drugs. See Muranishi, Crit.Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev.Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that theuptake and delivery of oligonucleotides, relatively complex moleculeswhich are known to be difficult to administer to animals and man, can begreatly improved even when administered by non-parenteral means throughthe use of a number of different classes of penetration enhancers.

In some embodiments, compositions for non-parenteral administrationinclude one or more modifications from naturally-occurringoligonucleotides (i.e. full-phosphodiester deoxyribosyl orfull-phosphodiester ribosyl oligonucleotides). Such modifications mayincrease binding affinity, nuclease stability, cell or tissuepermeability, tissue distribution, or other biological orpharmacokinetic property. Modifications may be made to the base, thelinker, or the sugar, in general, as discussed in more detail hereinwith regards to oligonucleotide chemistry. In some embodiments of theinvention, compositions for administration to a subject, and inparticular oral compositions for administration to an animal or humansubject, will comprise modified oligonucleotides having one or moremodifications for enhancing affinity, stability, tissue distribution, oranother biological property.

Suitable modified linkers include phosphorothioate linkers. In someembodiments according to the invention, the oligonucleotide has at leastone phosphorothioate linker. Phosphorothioate linkers provide nucleasestability as well as plasma protein binding characteristics to theoligonucleotide. Nuclease stability is useful for increasing the in vivolifetime of oligonucleotides, while plasma protein binding decreases therate of first pass clearance of oligonucleotide via renal excretion. Insome embodiments according to the present invention, the oligonucleotidehas at least two phosphorothioate linkers. In some embodiments, whereinthe oligonucleotide has exactly n nucleosides, the oligonucleotide hasfrom one to n−1 phosphorothioate linkages. In some embodiments, whereinthe oligonucleotide has exactly n nucleosides, the oligonucleotide hasn−1 phosphorothioate linkages. In other embodiments wherein theoligonucleotide has exactly n nucleoside, and n is even, theoligonucleotide has from 1 to n/2 phosphorothioate linkages, or, when nis odd, from 1 to (n−1)/2 phosphorothioate linkages. In someembodiments, the oligonucleotide has alternating phosphodiester (PO) andphosphorothioate (PS) linkages. In other embodiments, theoligonucleotide has at least one stretch of two or more consecutive POlinkages and at least one stretch of two or more PS linkages. In otherembodiments, the oligonucleotide has at least two stretches of POlinkages interrupted by at least on PS linkage.

In some embodiments, at least one of the nucleosides is modified on theribosyl sugar unit by a modification that imparts nuclease stability,binding affinity or some other beneficial biological property to thesugar. In some cases the sugar modification includes a 2′-modification,e.g. the 2′-OH of the ribosyl sugar is replaced or substituted. Suitablereplacements for 2′-OH include 2′-F and 2′-arabino-F. Suitablesubstitutions for OH include 2′-O-alkyl, e.g. 2-O-methyl, and2′-β-substituted alkyl, e.g. 2′-O-methoxyethyl, 2′-β-aminopropyl, etc.In some embodiments, the oligonucleotide contains at least one2′-modification. In some embodiments, the oligonucleotide contains atleast 2 2′-modifications. In some embodiments, the oligonucleotide hasat least one 2′-modification at each of the termini (i.e. the 3′- and5′-terminal nucleosides each have the same or different2′-modifications). In some embodiments, the oligonucleotide has at leasttwo sequential 2′-modifications at each end of the oligonucleotide. Insome embodiments, oligonucleotides further comprise at least onedeoxynucleoside. In particular embodiments, oligonucleotides comprise astretch of deoxynucleosides such that the stretch is capable ofactivating RNase (e.g. RNase H) cleavage of an RNA to which theoligonucleotide is capable of hybridizing. In some embodiments, astretch of deoxynucleosides capable of activating RNase-mediatedcleavage of RNA comprises about 6 to about 16, e.g. about 8 to about 16consecutive deoxynucleosides.

Oral compositions for administration of non-parenteral oligonucleotidecompositions of the present invention may be formulated in variousdosage forms such as, but not limited to, tablets, capsules, liquidsyrups, soft gels, suppositories, and enemas. The term “alimentarydelivery” encompasses e.g. oral, rectal, endoscopic andsublingual/buccal administration. A common requirement for these modesof administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of thenucleic acid(s) so administered.

Delivery of a drug via the oral mucosa, as in the case of buccal andsublingual administration, has several desirable features, including, inmany instances, a more rapid rise in plasma concentration of the drugthan via oral delivery (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711).

Endoscopy may be used for drug delivery directly to an interior portionof the alimentary tract. For example, endoscopic retrogradecystopancreatography (ERCP) takes advantage of extended gastroscopy andpermits selective access to the biliary tract and the pancreatic duct(Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591).Pharmaceutical compositions, including liposomal formulations, can bedelivered directly into portions of the alimentary canal, such as, e.g.,the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastricsubmucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) viaendoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs,1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington etal., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for directalimentary delivery of pharmaceutical compositions.

In some embodiments, oligonucleotide formulations may be administeredthrough the anus into the rectum or lower intestine. Rectalsuppositories, retention enemas or rectal catheters can be used for thispurpose and may be preferred when patient compliance might otherwise bedifficult to achieve (e.g., in pediatric and geriatric applications, orwhen the patient is vomiting or unconscious). Rectal administration canresult in more prompt and higher blood levels than the oral route.(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Becauseabout 50% of the drug that is absorbed from the rectum will bypass theliver, administration by this route significantly reduces the potentialfor first-pass metabolism (Benet et al., Chapter 1 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996).

One advantageous method of non-parenteral administration oligonucleotidecompositions is oral delivery. Some embodiments employ variouspenetration enhancers in order to effect transport of oligonucleotidesand other nucleic acids across mucosal and epithelial membranes.Penetration enhancers may be classified as belonging to one of fivebroad categories—surfactants, fatty acids, bile salts, chelating agents,and non-chelating non-surfactants (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92). Accordingly, someembodiments comprise oral oligonucleotide compositions comprising atleast one member of the group consisting of surfactants, fatty acids,bile salts, chelating agents, and non-chelating surfactants. Furtherembodiments comprise oral oligonucleotide comprising at least one fattyacid, e.g. capric or lauric acid, or combinations or salts thereof.Other embodiments comprise methods of enhancing the oral bioavailabilityof an oligonucleotide, the method comprising co-administering theoligonucleotide and at least one penetration enhancer.

Other excipients that may be added to oral oligonucleotide compositionsinclude surfactants (or “surface-active agents”), which are chemicalentities which, when dissolved in an aqueous solution, reduce thesurface tension of the solution or the interfacial tension between theaqueous solution and another liquid, with the result that absorption ofoligonucleotides through the alimentary mucosa and other epithelialmembranes is enhanced. In addition to bile salts and fatty acids,surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J.Pharm. Phamacol., 1988, 40, 252).

Fatty acids and their derivatives which act as penetration enhancers andmay be used in compositions of the present invention include, forexample, oleic acid, lauric acid, capric acid (n-decanoic acid),myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol),dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono-and di-glycerides thereof and/or physiologically acceptable saltsthereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate,linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

In some embodiments, oligonucleotide compositions for oral deliverycomprise at least two discrete phases, which phases may compriseparticles, capsules, gel-capsules, microspheres, etc. Each phase maycontain one or more oligonucleotides, penetration enhancers,surfactants, bioadhesives, effervescent agents, or other adjuvant,excipient or diluent. In some embodiments, one phase comprises at leastone oligonucleotide and at least one penetration enhancer. In someembodiments, a first phase comprises at least one oligonucleotide and atleast one penetration enhancer, while a second phase comprises at leastone penetration enhancer. In some embodiments, a first phase comprisesat least one oligonucleotide and at least one penetration enhancer,while a second phase comprises at least one penetration enhancer andsubstantially no oligonucleotide. In some embodiments, at least onephase is compounded with at least one degradation retardant, such as acoating or a matrix, which delays release of the contents of that phase.In some embodiments, a first phase comprises at least oneoligonucleotide, and at least one penetration enhancer, while a secondphase comprises at least one penetration enhancer and arelease-retardant. In particular embodiments, an oral oligonucleotidecomprises a first phase comprising particles containing anoligonucleotide and a penetration enhancer, and a second phasecomprising particles coated with a release-retarding agent andcontaining penetration enhancer.

A variety of bile salts also function as penetration enhancers tofacilitate the uptake and bioavailability of drugs. The physiologicalroles of bile include the facilitation of dispersion and absorption oflipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus, the term “bile salt” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579).

In some embodiments, penetration enhancers useful in some embodiments ofpresent invention are mixtures of penetration enhancing compounds. Onesuch penetration enhancer is a mixture of UDCA (and/or CDCA) with capricand/or lauric acids or salts thereof e.g. sodium. Such mixtures areuseful for enhancing the delivery of biologically active substancesacross mucosal membranes, in particular intestinal mucosa. Otherpenetration enhancer mixtures comprise about 5-95% of bile acid orsalt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid.Particular penetration enhancers are mixtures of the sodium salts ofUDCA, capric acid and lauric acid in a ratio of about 1:2:2respectively. Another such penetration enhancer is a mixture of capricand lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (molebasis). In particular embodiments capric acid and lauric acid arepresent in molar ratios of e.g. about 0.1:1 to about 1:0.1, inparticular about 0.5:1 to about 1:0.5.

Other excipients include chelating agents, i.e. compounds that removemetallic ions from solution by forming complexes therewith, with theresult that absorption of oligonucleotides through the alimentary andother mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315).Chelating agents of the invention include, but are not limited to,disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. ControlRel., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers maybe defined as compounds that demonstrate insignificant activity aschelating agents or as surfactants but that nonetheless enhanceabsorption of oligonucleotides through the alimentary and other mucosalmembranes (Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1). This class of penetration enhancers includes, butis not limited to, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), can be used.

Some oral oligonucleotide compositions also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which may beinert (i.e., does not possess biological activity per se) or may benecessary for transport, recognition or pathway activation or mediation,or is recognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4-tisothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6, 177).

A “pharmaceutical carrier” or “excipient” may be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal. Theexcipient may be liquid or solid, and is selected with the plannedmanner of administration in mind so as to provide for the desired bulk,consistency, etc., when combined with a nucleic acid and the othercomponents of a given pharmaceutical composition. Typical pharmaceuticalcarriers include, but are not limited to, binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.); fillers (e.g., lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB™disintegrating agent); and wetting agents (e.g., sodium lauryl sulphate,etc.).

Oral oligonucleotide compositions may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipuritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. For example, the first targetmay be an apolipoprotein(a) target, and the second target may be aregion from another nucleotide sequence. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same apolipoprotein(a) nucleic acid target.Numerous examples of antisense compounds are illustrated herein, andothers may be selected from among suitable compounds known in the art.Two or more combined compounds may be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, from 0.1 μg to 10 g per kg of bodyweight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mgper kg of body weight, from 100 μg to 10 mg per kg of body weight, orfrom 1 mg to 5 mg per kg of body weight, and may be given once or moredaily, weekly, monthly or yearly, or even once every 2 to 20 years.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state, wherein the oligonucleotideis administered in maintenance doses, ranging from 0.01 μg to 100 g perkg of body weight, once or more daily, to once every 20 years.

The effects of treatments with therapeutic compositions can be assessedfollowing collection of tissues or fluids from a patient or subjectreceiving said treatments. It is known in the art that a biopsy samplecan be procured from certain tissues without resulting in detrimentaleffects to a patient or subject. In certain embodiments, a tissue andits constituent cells comprise, but are not limited to, blood (e.g.,hematopoietic cells, such as human hematopoietic progenitor cells, humanhematopoietic stem cells, CD34⁺ cells CD4⁺ cells), lymphocytes and otherblood lineage cells, bone marrow, breast, cervix, colon, esophagus,lymph node, muscle, peripheral blood, oral mucosa and skin. In otherembodiments, a fluid and its constituent cells comprise, but are notlimited to, blood, urine, semen, synovial fluid, lymphatic fluid andcerebro-spinal fluid. Tissues or fluids procured from patients can beevaluated for expression levels of the target mRNA or protein.Additionally, the mRNA or protein expression levels of other genes knownor suspected to be associated with the specific disease state, conditionor phenotype can be assessed. mRNA levels can be measured or evaluatedby real-time PCR, Northern blot, in situ hybridization or DNA arrayanalysis. Protein levels can be measured or evaluated by ELISA,immunoblotting, quantitative protein assays, protein activity assays(for example, caspase activity assays) immunohistochemistry orimmunocytochemistry. Furthermore, the effects of treatment can beassessed by measuring biomarkers associated with the disease orcondition in the aforementioned tissues and fluids, collected from apatient or subject receiving treatment, by routine clinical methodsknown in the art. These biomarkers include but are not limited to:glucose, cholesterol, lipoproteins, triglycerides, free fatty acids andother markers of glucose and lipid metabolism; liver transaminases,bilirubin, albumin, blood urea nitrogen, creatine and other markers ofkidney and liver function; interleukins, tumor necrosis factors,intracellular adhesion molecules, C-reactive protein and other markersof inflammation; testosterone, estrogen and other hormones; tumormarkers; vitamins, minerals and electrolytes.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same. Each of the references, GENBANK® accession numbers, aswell as each application from which the present application claimspriority, and the like recited in the present application isincorporated herein by reference in its entirety.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and InternationalPatent Publication No. WO 02/36743; 5′-O-Dimethoxytrityl-thymidineintermediate for 5-methyl dC amidite,51-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-β-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors, including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inInternational Patent Application Nos. PCT/US94/00902 and PCT/US93/06976(published as International Patent Publication Nos. WO 94/17093 and WO94/02499, respectively), herein incorporated by reference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825; 5,386,023; 5,489,677; 5,602,240; and 5,610,289; all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group that has the following important properties.It is stable to the conditions of nucleoside phosphoramidite synthesisand oligonucleotide synthesis. However, after oligonucleotide synthesisthe oligonucleotide is treated with methylamine, which not only cleavesthe oligonucleotide from the solid support but also removes the acetylgroups from the orthoesters. The resulting 2-ethyl-hydroxyl substituentson the orthoester are less electron withdrawing than the acetylatedprecursor. As a result, the modified orthoester becomes more labile toacid-catalyzed hydrolysis. Specifically, the rate of cleavage isapproximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 μM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspectrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry).

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/5 oligonucleosides are synthesized accordingto U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingApolipoprotein(a)

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target apolipoprotein(a). Thenucleobase sequence of the antisense strand of the duplex comprises atleast an 8-nucleobase portion of an oligonucleotide in Table 1. The endsof the strands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini. The antisense and sense strands of theduplex comprise from about 17 to 25 nucleotides, or from about 19 to 23nucleotides. Alternatively, the antisense and sense strands comprise 20,21 or 22 nucleotides.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 97) and having a two-nucleobase overhangof deoxythymidine(dT) has the following structure (Antisense SEQ ID NO:98, Complement SEQ ID NO: 99):

Overhangs can range from 2 to 6 nucleobases and these nucleobases may ormay not be complementary to the target nucleic acid. In anotherembodiment, the duplexes may have an overhang on only one terminus.

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 97) is prepared withblunt ends (no single stranded overhang) as shown (Antisense SEQ ID NO:97, Complement SEQ ID NO: 100):

The RNA duplex can be unimolecular or bimolecular; i.e., the two strandscan be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 15 μL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate apolipoprotein(a) expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN™ reagent (Invitrogen Life Technologies, Carlsbad, Calif.) andthe desired duplex antisense compound at a final concentration of 200nM. After 5 hours of treatment, the medium is replaced with freshmedium. Cells are harvested 16 hours after treatment, at which time RNAis isolated and target reduction measured by RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full-length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis were determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis-96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQapparatus) or, for individually prepared samples, on a commercial CEapparatus (e.g., Beckman P/ACE™ 5000, ABI 270 apparatus). Base andbackbone composition was confirmed by mass analysis of the compoundsutilizing electrospray-mass spectroscopy. All assay test plates werediluted from the master plate using single and multi-channel roboticpipettors. Plates were judged to be acceptable if at least 85% of thecompounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effects of antisense compounds on target nucleic acid expression aretested in any of a variety of cell types, provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 μg/mL (Invitrogen Corporation,Carlsbad, Calif.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence. Cells were seeded into96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/wellfor use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 μg/mL (Invitrogen Corporation, Carlsbad, Calif.). Cellswere routinely passaged by trypsinization and dilution when they reached90% confluence.

NHDF Cells:

Human neonatal dermal fibroblasts (NHDFs) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 medium containing 3.75 μg/mL LIPOFECTIN™ reagent (InvitrogenCorporation, Carlsbad, Calif.) and the desired concentration ofoligonucleotide. Cells are treated and data are obtained in triplicate.After 4-7 hours of treatment at 37° C., the medium was replaced withfresh medium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of Apolipoprotein(a)Expression

Antisense modulation of apolipoprotein(a) expression can be assayed in avariety of ways known in the art. For example, apolipoprotein(a) mRNAlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or poly(A)+ mRNA. The preferred method of RNAanalysis of the present invention is the use of total cellular RNA asdescribed in other examples herein. Methods of RNA isolation are wellknown in the art. Northern blot analysis is also routine in the art.Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of apolipoprotein(a) can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toapolipoprotein(a) can be identified and obtained from a variety ofsources, such as the MSRS catalog of antibodies (Aerie Corporation,Birmingham, Mich.), or can be prepared via conventional monoclonal orpolyclonal antibody generation methods well known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof Apolipoprotein(a) Inhibitors Phenotypic Assays

Once apolipoprotein(a) inhibitors have been identified by the methodsdisclosed herein, the compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of apolipoprotein(a) in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated withapolipoprotein(a) inhibitors identified from the in vitro studies aswell as control compounds at optimal concentrations which are determinedby the methods described above. At the end of the treatment period,treated and untreated cells are analyzed by one or more methods specificfor the assay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status, which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the genotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the apolipoprotein(a)inhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

The cells subjected to the phenotypic assays described herein derivefrom in vitro cultures or from tissues or fluids isolated from livingorganisms, both human and non-human. In certain embodiments, a tissueand its constituent cells comprise, but are not limited to, blood (e.g.,hematopoietic cells, such as human hematopoietic progenitor cells, humanhematopoietic stem cells, CD34⁺ cells CD4⁺ cells), lymphocytes and otherblood lineage cells, bone marrow, brain, stem cells, blood vessel,liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryonic,endometrium, endothelial, epithelial, esophagus, facia, fibroblast,follicular, ganglion cells, glial cells, goblet cells, kidney, lymphnode, muscle, neuron, ovaries, pancreas, peripheral blood, prostate,skin, skin, small intestine, spleen, stomach, testes and fetal tissue.In other embodiments, a fluid and its constituent cells comprise, but isnot limited to, blood, urine, synovial fluid, lymphatic fluid andcerebro-spinal fluid. The phenotypic assays may also be performed ontissues treated with apolipoprotein(a) inhibitors ex vivo.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, including humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study.

To account for the psychological effects of receiving treatments,volunteers are randomly given placebo or apolipoprotein(a) inhibitor.Furthermore, to prevent the doctors from being biased in treatments,they are not informed as to whether the medication they areadministering is a apolipoprotein(a) inhibitor or a placebo. Using thisrandomization approach, each volunteer has the same chance of beinggiven either the new treatment or the placebo.

Volunteers receive either the apolipoprotein(a) inhibitor or placebo foreight week period with biological parameters associated with theindicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingapolipoprotein(a) or apolipoprotein(a) protein levels in body fluids,tissues or organs compared to pre-treatment levels. Other measurementsinclude, but are not limited to, indices of the disease state orcondition being treated, body weight, blood pressure, serum titers ofpharmacologic indicators of disease or toxicity as well as ADME(absorption, distribution, metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and apolipoprotein(a) inhibitor treatment. Ingeneral, the volunteers treated with placebo have little or no responseto treatment, whereas the volunteers treated with the apolipoprotein(a)inhibitor show positive trends in their disease state or condition indexat the conclusion of the study.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY™ 96 kit and buffers purchasedfrom Qiagen, Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY™ 96 well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY™ 96 plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY™ 96plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY™ 96 plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNase free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN® Bio-Robot™ 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of Apolipoprotein(a) mRNALevels

Quantitation of apolipoprotein(a) mRNA levels was accomplished byreal-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ Sequence Detection System. In each assay, aseries of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

Prior to the real-time PCR, isolated RNA is subjected to a reversetranscriptase (RT) reaction, for the purpose of generating complementaryDNA (cDNA), from which the real-time PCR product is amplified. Reversetranscriptase and PCR reagents were obtained from InvitrogenCorporation, (Carlsbad, Calif.). RT, real-time PCR reactions carried outby adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂,375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primerand reverse primer, 125 nM of probe, 4 Units RNase inhibitor, 1.25 UnitsPLATINUM® Taq polymerase, 5 Units MuLV reverse transcriptase, and2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution(20-200 ng). The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe PLATINUM® Taq polymerase, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension). The method of obtaining gene targetquantities by RT, real-time PCR is herein referred to as real-time PCR.

Gene target quantities obtained by RT, real-time PCR are normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RIBOGREEN™ reagent(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real-time PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RIBOGREEN™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN™ reagent are taught in Jones,L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 apparatus (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

Probes and primers to human apolipoprotein(a) were designed to hybridizeto a human apolipoprotein(a) sequence, using published sequenceinformation (GENBANK® accession number NM_(—)005577.1, incorporatedherein as SEQ ID NO: 4). For human apolipoprotein(a) the PCR primerswere:

forward primer: CAGCTCCTTATTGTTATACGAGGGA (SEQ ID NO: 5)reverse primer: TGCGTCTGAGCATTGCGT (SEQ ID NO: 6) and thePCR probe was: FAM-CCCGGTGTCAGGTGGGAGTACTGC-TAMRA (SEQ ID NO: 7) whereFAM is the fluorescent dye and TAMRA is the quencher dye.

Gene target quantities in mouse cells are tissues are normalized usingmouse GAPDH expression. For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 8)reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 9) andthe PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ IDNO: 10) where JOE is the fluorescent reporter dye and TAMRA is thequencher dye.

Example 14 Northern Blot Analysis of Apolipoprotein(a) mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ reagent (TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% agarose gels containing1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes(Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillarytransfer using a Northern/Southern Transfer buffer system (TEL-TEST “B”Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER™ UV Crosslinker 2400 apparatus (Stratagene, Inc, La Jolla,Calif.) and then probed using QUICKHYB™ hybridization solution(Stratagene, La Jolla, Calif.) using manufacturer's recommendations forstringent conditions.

To detect human apolipoprotein(a), a human apolipoprotein(a) specificprobe was prepared by PCR using the forward primerCAGCTCCTTATTGTTATACGAGGGA (SEQ ID NO: 5) and the reverse primerTGCGTCTGAGCATTGCGT (SEQ ID NO: 6). To normalize for variations inloading and transfer efficiency membranes were stripped and probed forhuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ apparatus and IMAGEQUANT™ Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

Example 15 Antisense Inhibition of Human Apolipoprotein(a) Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humanapolipoprotein(a) RNA, using published sequences (GENBANK® accessionnumber NM_(—)005577.1, incorporated herein as SEQ ID NO: 4). Thecompounds are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Apolipoprotein(a) is found in humans, nonhuman primates and the Europeanhedgehog, but not in common laboratory animals such as rats and mice.Transgenic mice which express human apolipoprotein(a) have beenengineered (Chiesa et al., J. Biol. Chem., 1992, 267, 24369-24374). Theuse of primary hepatocytes prepared from human apolipoprotein(a)transgenic mice circumvents the issue of variability when testingantisense oligonucleotide activity in primary cells. Accordingly,primary mouse hepatocytes prepared from the human apolipoprotein(a)transgenic mice were used to investigate the effects of antisenseoligonucleotides on human apolipoprotein(a) expression. The humanapolipoprotein(a) transgenic mice were obtained from Dr. Robert Pitasand Dr. Matthias Schneider in the Gladstone Institute at the Universityof California, San Francisco. Primary hepatocytes were isolated fromthese mice and were cultured in DMEM, high glucose (InvitrogenCorporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum,(Invitrogen Corporation, Carlsbad, Calif.), 100 units per mL penicillinand 100 μg/mL streptomycin (Invitrogen Corporation, Carlsbad, Calif.).For treatment with oligonucleotide, cells were washed once withserum-free DMEM and subsequently transfected with a dose of 150 nM ofantisense oligonucleotide using LIPOFECTIN™ reagent (InvitrogenCorporation, Carlsbad, Calif.) as described in other examples herein.The compounds were analyzed for their effect on human apolipoprotein(a)mRNA levels by quantitative real-time PCR as described in other examplesherein. Gene target quantities obtained by real time RT-PCR werenormalized using mouse GAPDH.

Data are averages from three experiments in which primary transgenicmouse hepatocytes were treated with 150 nM of antisense oligonucleotidestargeted to human apolipoprotein(a).

TABLE 1 Inhibition of human apolipoprotein(a) mRNA levelsby chimeric phosphorothioate oligonucleotideshaving 2′-MOE wings and a deoxy gap TAR- GET SEQ TAR- SEQ ID GET % IDISIS # REGION NO SITE SEQUENCE INHIB NO 144367 Coding 4 174ggcaggtccttcctgtgaca 53 11 144368 Coding 4 352 tctgcgtctgagcattgcgt 8712 144369 Coding 4 522 aagcttggcaggttcttcct 0 13 144370 Coding 4 1743tcggaggcgcgacggcagtc 40 14 144371 Coding 4 2768 cggaggcgcgacggcagtcc 015 144372 Coding 4 2910 ggcaggttcttcctgtgaca 65 16 144373 Coding 4 3371ataacaataaggagctgcca 50 17 144374 Coding 4 4972 gaccaagcttggcaggttct 6218 144375 Coding 4 5080 taacaataaggagctgccac 36 19 144376 Coding 4 5315tgaccaagcttggcaggttc 25 20 144377 Coding 4 5825 ttctgcgtctgagcattgcg 3821 144378 Coding 4 6447 aacaataaggagctgccaca 29 22 144379 Coding 4 7155acctgacaccgggatccctc 79 23 144380 Coding 4 7185 ctgagcattgcgtcaggttg 1624 144381 Coding 4 8463 agtagttcatgatcaagcca 71 25 144382 Coding 4 8915gacggcagtcccttctgcgt 34 26 144383 Coding 4 9066 ggcaggttcttccagtgaca 527 144384 Coding 4 10787 tgaccaagcttggcaagttc 31 28 144385 Coding 411238 tataacaccaaggactaatc 9 29 144386 Coding 4 11261ccatctgacattgggatcca 66 30 144387 Coding 4 11461 tgtggtgtcatagaggacca 3631 144388 Coding 4 11823 atgggatcctccgatgccaa 55 32 144389 Coding 411894 acaccaagggcgaatctcag 58 33 144390 Coding 4 11957ttctgtcactggacatcgtg 59 34 144391 Coding 4 12255 cacacggatcggttgtgtaa 5835 144392 Coding 4 12461 acatgtccttcctgtgacag 51 36 144393 Coding 412699 cagaaggaggccctaggctt 33 37 144394 Coding 4 13354ctggcggtgaccatgtagtc 52 38 144395 3′UTR 4 13711 tctaagtaggttgatgcttc 6839 144396 3′UTR 4 13731 tccttacccacgtttcagct 70 40 144397 3′UTR 4 13780ggaacagtgtcttcgtttga 63 41 144398 3′UTR 4 13801 gtttggcatagctggtagct 4442 144399 3′UTR 4 13841 accttaaaagcttatacaca 57 43 144400 3′UTR 4 13861atacagaatttgtcagtcag 21 44 144401 3′UTR 4 13881 gtcatagctatgacacctta 4645

As shown in Table 1, SEQ ID NOs 11, 12, 14, 16, 17, 18, 19, 21, 23, 25,30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43 and 45 demonstratedat least 35% inhibition of human apolipoprotein(a) expression in thisassay and are therefore preferred. More preferred are SEQ ID NOs 23, 12and 40. The target regions to which these preferred sequences arecomplementary are herein referred to as “preferred target segments” andare therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in Table 2. Thesesequences are shown to contain thymine (T) but one of skill in the artwill appreciate that thymine (T) is generally replaced by uracil (U) inRNA sequences. The sequences represent the reverse complement of thepreferred antisense compounds shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds. Also shown in Table 2 is thespecies in which each of the preferred target segments was found.

TABLE 2 Sequence and position of preferred target segmentsidentified in apolipoprotein(a). TARGET REV SITE SEQ ID TARGET COMP OFSEQ ID ID NO SITE SEQUENCE SEQ ID ACTIVE IN NO 57364 4 174tgtcacaggaaggacctgcc 11 H. sapiens 46 57365 4 352 acgcaatgctcagacgcaga12 H. sapiens 47 57367 4 1743 gactgccgtcgcgcctccga 14 H. sapiens 4857369 4 2910 tgtcacaggaagaacctgcc 16 H. sapiens 49 57370 4 3371tggcagctccttattgttat 17 H. sapiens 50 57371 4 4972 agaacctgccaagcttggtc18 H. sapiens 51 57372 4 5080 gtggcagctccttattgtta 19 H. sapiens 5257374 4 5825 cgcaatgctcagacgcagaa 21 H. sapiens 53 57376 4 7155gagggatcccggtgtcaggt 23 H. sapiens 54 57378 4 8463 tggcttgatcatgaactact25 H. sapiens 55 57383 4 11261 tggatcccaatgtcagatgg 30 H. sapiens 5657384 4 11461 tggtcctctatgacaccaca 31 H. sapiens 57 57385 4 11823ttggcatcggaggatcccat 32 H. sapiens 58 57386 4 11894 ctgagattcgcccttggtgt33 H. sapiens 59 57387 4 11957 cacgatgtccagtgacagaa 34 H. sapiens 6057388 4 12255 ttacacaaccgatccgtgtg 35 H. sapiens 61 57389 4 12461ctgtcacaggaaggacatgt 36 H. sapiens 62 57391 4 13354 gactacatggtcaccgccag38 H. sapiens 63 57392 4 13711 gaagcatcaacctacttaga 39 H. sapiens 6457393 4 13731 agctgaaacgtgggtaagga 40 H. sapiens 65 57394 4 13780tcaaacgaagacactgttcc 41 H. sapiens 66 57395 4 13801 agctaccagctatgccaaac42 H. sapiens 67 57396 4 13841 tgtgtataagcttttaaggt 43 H. sapiens 6857398 4 13881 taaggtgtcatagctatgac 45 H. sapiens 69

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of apolipoprotein(a).

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, siRNAs,external guide sequence (EGS) oligonucleotides, alternate splicers, andother short oligomeric compounds that hybridize to at least a portion ofthe target nucleic acid.

Example 16 Western Blot Analysis of Apolipoprotein(a) Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μl/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to apolipoprotein(a) isused, with a radiolabeled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands are visualizedusing a PHOSPHORIMAGER™ apparatus (Molecular Dynamics, SunnyvaleCalif.).

Example 17 Antisense Inhibition of Human Apolipoprotein(a) in TransgenicPrimary Mouse Hepatocytes: Dose Response

In accordance with the present invention, antisense oligonucleotidesidentified as having good activity based on the results in Example 15were further investigated in dose-response studies. Primary hepatocytesfrom human apolipoprotein(a) transgenic mice were treated with 10, 50,150 or 300 nM of ISIS 144396 (SEQ ID NO: 40), ISIS 144368 (SEQ ID NO:12), ISIS 144379 (SEQ ID NO: 23) or ISIS 113529 (CTCTTACTGTGCTGTGGACA,SEQ ID NO: 70). ISIS 113529, which does not target apolipoprotein(a),was used as a control oligonucleotide and is a chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-methoxyethyl (2′-MOE)nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All cytidine residues are 5-methylcytidines.

Following 24 hours of exposure to antisense oligonucleotides, targetmRNA expression levels were evaluated by quantitative real-time PCR asdescribed in other examples herein. The results are the average of 4experiments for apolipoprotein(a) antisense oligonucleotides and theaverage of 12 experiments for the control oligonucleotide. The data areexpressed as percent inhibition of apolipoprotein(a) expression relativeto untreated controls and are shown in Table 3.

TABLE 3 Antisense inhibition of human apolipoprotein(a) in transgenicprimary mouse hepatocytes: dose response % Inhibition of transgenichuman lipoprotein(a) ISIS # Oligonucleotide dose 144396 144368 144379113529  10 nM 0 11 55 N.D.  50 nM 0 26 73 N.D. 150 nM 0 58 85 N.D. 300nM 9 62 89 0

These data demonstrate that ISIS 144368 and ISIS 144379 inhibited theexpression of human apolipoprotein(a) in a dose-dependent fashion.

Example 18 Oil Red O Stain

Hepatic steatosis, or accumulation of lipids in the liver, is assessedby routine histological analysis of frozen liver tissue sections stainedwith oil red 0 stain, which is commonly used to visualize lipiddeposits, and counterstained with hematoxylin and eosin, to visualizenuclei and cytoplasm, respectively. Tissue is preserved in 10%neutral-buffered formalin, embedded in paraffin, sectioned and stained.

Example 19 Animal Models

In addition to human systems, which express apolipoprotein(a),biological systems of other mammals are also available for studies ofexpression products of the LPA gene as well as for studies of the Lp(a)particles and their role in physiologic processes.

Transgenic mice which express human apolipoprotein(a) have beenengineered (Chiesa et al., J. Biol. Chem., 1992, 267, 24369-24374) andare used as an animal model for the investigation of the in vivoactivity of the oligonucleotides of this invention. Although transgenicmice expressing human apolipoprotein(a) exist, they fail to assembleLp(a) particles because of the inability of human apolipoprotein(a) toassociate with mouse apolipoprotein B. When mice expressing humanapolipoprotein(a) are bred to mice expressing human apolipoprotein B,the Lp(a) particle is efficiently assembled (Callow et al., Proc. Natl.Acad. Sci. USA, 1994, 91, 2130-2134). Accordingly mice expressing bothhuman apolipoprotein(a) and human apolipoprotein B transgenes are usedfor animal model studies in which the secretion of the Lp(a) particle isevaluated.

Where additional genetic alterations are necessary, mice with either asingle human transgene (human apolipoprotein(a) or human apolipoproteinB) or both human transgenes (human apolipoprotein(a) and humanapolipoprotein B) are bred to mice with a desired genetic mutation. Theoffspring with the desired combination of transgene(s) and geneticmutation(s) is selected for use as an animal model. In one nonlimitingexample, mice expressing both human apolipoprotein(a) and humanapolipoprotein B are bred to mice with a mutation in the leptin gene,yielding offspring producing human Lp(a) particles in an ob/ob model ofobesity and diabetes.

ob/ob Mice

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. ob/ob mice have a mutation in the leptin gene which resultsin obesity and hyperglycemia. As such, these mice are a useful model forthe investigation of obesity and treatments designed to reduce obesity.

Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) are fed a diet with a fat content of 10-15% and aresubcutaneously injected with oligonucleotides of the present inventionor a control oligonucleotide at a dose of 5, 10 or 25 mg/kg two timesper week for 4 weeks. Saline-injected animals and leptin wildtypelittermates (i.e. lean littermates) serve as controls. After thetreatment period, mice are sacrificed and target levels are evaluated inliver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNAisolation and target mRNA expression level quantitation are performed asdescribed by other examples herein.

To assess the physiological effects resulting from antisense inhibitionof target apolipoprotein(a) mRNA, the ob/ob mice that receive antisenseoligonucleotide treatment are further evaluated at the end of thetreatment period for serum lipids, serum apolipoproteins, serum freefatty acids, serum cholesterol (CHOL), liver triglycerides, and fattissue triglycerides. Serum components are measured on routine clinicaldiagnostic instruments. Tissue triglycerides are extracted using anacetone extraction technique known in the art, and subsequently measuredby ELISA. The presence of the Lp(a) particle in the serum is measuredusing a commercially available ELISA kit (ALerCHEK Inc., Portland, Me.).Hepatic steatosis, or accumulation of lipids in the liver, is assessedby measuring the liver triglyceride content. Hepatic steatosis is alsoassessed by routine histological analysis of frozen liver tissuesections stained with oil red O stain, which is commonly used tovisualize lipid deposits, and counterstained with hematoxylin and eosin,to visualize nuclei and cytoplasm, respectively.

The effects of apolipoprotein(a) inhibition on glucose and insulinmetabolism are also evaluated in the ob/ob mice treated with antisenseoligonucleotides of this invention. Plasma glucose is measured at thestart of the antisense oligonucleotide treatment and after 2 weeks and 4weeks of treatment. Plasma insulin is similarly at the beginning to ofthe treatment, and following 2 weeks and 4 weeks of treatment. Glucoseand insulin tolerance tests are also administered in fed and fastedmice. Mice receive intraperitoneal injections of either glucose orinsulin, and the blood glucose and insulin levels are measured beforethe insulin or glucose challenge and at 15, 20 or 30 minute intervalsfor up to 3 hours.

To assess the metabolic rate of ob/ob mice treated with antisenseoligonucleotides of this invention, the respiratory quotient and oxygenconsumption of the mice are also measured.

The ob/ob mice that received antisense oligonucleotide treatment arefurther evaluated at the end of the treatment period for the effects ofapolipoprotein(a) inhibition on the expression of genes that participatein lipid metabolism, cholesterol biosynthesis, fatty acid oxidation,fatty acid storage, gluconeogenesis and glucose metabolism. These genesinclude, but are not limited to, HMG-CoA reductase, acetyl-CoAcarboxylase 1 and acetyl-CoA carboxylase 2, carnitinepalmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphataseand phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormonesensitive lipase. mRNA levels in liver and white and brown adiposetissue are quantitated by real-time PCR as described in other examplesherein, employing primer-probe sets that were generated using publishedsequences of each gene of interest.

db/db Mice

A deficiency in the leptin hormone receptor mouse also results inobesity and hyperglycemia. These mice are referred to as db/db mice and,like the ob/ob mice, are used as a mouse model of obesity.

Seven-week old male C57Bl/6J-Lepr db/db mice (Jackson Laboratory, BarHarbor, Me.) are fed a diet with a fat content of 15-20% and aresubcutaneously injected with oligonucleotides of this invention or acontrol oligonucleotide at a dose of 5, 10 or 25 mg/kg two times perweek for 4 weeks. Saline-injected animals and leptin receptor wildtypelittermates (i.e. lean littermates) serve as controls. After thetreatment period, mice are sacrificed and apolipoprotein(a) levels areevaluated in liver, brown adipose tissue (BAT) and white adipose tissue(WAT). RNA isolation and apolipoprotein(a) mRNA expression levelquantitation are performed as described by other examples herein.

After the treatment period, mice are sacrificed and apolipoprotein(a)levels are evaluated in liver, brown adipose tissue (BAT) and whiteadipose tissue (WAT). RNA isolation and apolipoprotein(a) mRNAexpression level quantitation are performed as described by otherexamples herein.

To assess the physiological effects resulting from antisense inhibitionof apolipoprotein(a) mRNA, the db/db mice that receive antisenseoligonucleotide treatment are further evaluated at the end of thetreatment period for serum lipids, serum apolipoproeins, serum freefatty acids, serum cholesterol (CHOL), liver triglycerides, and fattissue triglycerides. Serum components are measured on routine clinicaldiagnostic instruments. Tissue triglycerides are extracted using anacetone extraction technique known in the art, and subsequently measuredby ELISA. The presence of the Lp(a) particle in the serum is measuredusing a commercially available ELISA kit (ALerCHEK Inc., Portland, Me.).Hepatic steatosis, or accumulation of lipids in the liver, is assessedby measuring the liver triglyceride content. Hepatic steatosis is alsoassessed by routine histological analysis of frozen liver tissuesections stained with oil red O stain, which is commonly used tovisualize lipid deposits, and counterstained with hematoxylin and eosin,to visualize nuclei and cytoplasm, respectively.

The effects of apolipoprotein(a) inhibition on glucose and insulinmetabolism are also evaluated in the db/db mice treated with antisenseoligonucleotides. Plasma glucose is measured at the start of theantisense oligonucleotide treatment and after 2 weeks and 4 weeks oftreatment. Plasma insulin is similarly at the beginning to of thetreatment, and following 2 weeks and 4 weeks of treatment. Glucose andinsulin tolerance tests are also administered in fed and fasted mice.Mice receive intraperitoneal injections of either glucose or insulin,and the blood glucose levels are measured before the insulin or glucosechallenge and 15, 30, 60, 90 and 120 minutes following the injection.

To assess the metabolic rates of db/db mice treated with antisenseoligonucleotides, the respiratory quotients and oxygen consumptions ofthe mice are also measured.

The db/db mice that received antisense oligonucleotide treatment arefurther evaluated at the end of the treatment period for the effects ofapolipoprotein(a) inhibition on the expression of genes that participatein lipid metabolism, cholesterol biosynthesis, fatty acid oxidation,fatty acid storage, gluconeogenesis and glucose metabolism. These genesinclude, but are not limited to, HMG-CoA reductase, acetyl-CoAcarboxylase 1 and acetyl-CoA carboxylase 2, carnitinepalmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphataseand phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormonesensitive lipase. mRNA levels in liver and white and brown adiposetissue are quantitated by real-time PCR as described in other examplesherein, employing primer-probe sets that were generated using publishedsequences of each gene of interest.

Lean Mice

C57Bl/6 mice are maintained on a standard rodent diet and are used ascontrol (lean) animals. Seven-week old male C57Bl/6 mice are fed a dietwith a fat content of 4% and are subcutaneously injected witholigonucleotides of this invention or control oligonucleotide at a doseof 5, 10 or 25 mg/kg two times per week for 4 weeks. Saline-injectedanimals serve as a control. After the treatment period, mice aresacrificed and apolipoprotein(a) levels are evaluated in liver, brownadipose tissue (BAT) and white adipose tissue (WAT). RNA isolation andapolipoprotein(a) mRNA expression level quantitation are performed asdescribed by other examples herein.

To assess the physiological effects resulting from antisense inhibitionof apolipoprotein(a) mRNA, the lean mice that receive antisenseoligonucleotide treatment are further evaluated at the end of thetreatment period for serum lipids, serum free fatty acids, serumcholesterol (CHOL), liver triglycerides, and fat tissue triglycerides.Serum components are measured on routine clinical diagnosticinstruments. Tissue triglycerides are extracted using an acetoneextraction technique known in the art, and subsequently measured byELISA. The presence of the Lp(a) particle in the serum is measured usinga commercially available ELISA kit (ALerCHEK Inc., Portland, Me.).Hepatic steatosis, i.e. accumulation of lipids in the liver, is assessedby measuring the liver triglyceride content. Hepatic steatosis is alsoassessed by routine histological analysis of frozen liver tissuesections stained with oil red O stain, which is commonly used tovisualize lipid deposits, and counterstained with hematoxylin and eosin,to visualize nuclei and cytoplasm, respectively.

The effects of apolipoprotein(a) inhibition on glucose and insulinmetabolism are also evaluated in the lean mice treated with antisenseoligonucleotides of this invention. Plasma glucose is measured at thestart of the antisense oligonucleotide treatment and after 2 weeks and 4weeks of treatment. Plasma insulin is similarly at the beginning to ofthe treatment, and following 2 weeks and 4 weeks of treatment. Glucoseand insulin tolerance tests are also administered in fed and fastedmice. Mice receive intraperitoneal injections of either glucose orinsulin, and the blood glucose levels are measured before the insulin orglucose challenge and 15, 30, 60, 90 and 120 minutes following theinjection.

To assess the metabolic rates of lean mice treated with antisenseoligonucleotides of this invention, the respiratory quotients and oxygenconsumptions of the mice can also be measured.

The lean mice that received antisense oligonucleotide treatment arefurther evaluated at the end of the treatment period for the effects ofapolipoprotein(a) inhibition on the expression of genes that participatein lipid metabolism, cholesoterol biosynthesis, fatty acid oxidation,fatty acid storage, gluconeogenesis and glucose metabolism. These genesinclude, but are not limited to, HMG-CoA reductase, acetyl-CoAcarboxylase 1 and acetyl-CoA carboxylase 2, carnitinepalmitoyltransferase I and glycogen expressing both humanapolipoprotein(a) and human apolipoprotein B are bred to mice with amutation in the leptin gene, yielding offspring producing human Lp(a)particles in an ob/ob model of obesity and diabetes.

ob/ob Mice

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. ob/ob mice have a mutation in the leptin gene which resultsin obesity and hyperglycemia. As such, these mice are a useful model forthe investigation of obesity and treatments designed to reduce obesity.

Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) are fed a diet with a fat content of 10-15% and aresubcutaneously injected with oligonucleotides of the present inventionor a control oligonucleotide at a dose of 5, 10 or 25 mg/kg two timesper week for 4 weeks. Saline-injected animals and leptin wildtypelittermates (i.e. lean littermates) serve as controls. After thetreatment period, mice are sacrificed and target levels are evaluated inliver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNAisolation and target mRNA expression level quantitation are performed asdescribed by other examples herein.

To assess the physiological effects resulting from antisense inhibitionof target apolipoprotein(a) mRNA, the ob/ob mice that receive antisenseoligonucleotide treatment are further evaluated at the end of thetreatment period for serum lipids, serum apolipoproteins, serum freefatty acids, serum cholesterol (CHOL), liver reverse primer:ACAGGGCTTTTCTCAGGTGGT (SEQ ID NO: 72) and the additional PCR probe was:FAM-CCAAGCACAGAGGCTCCTTCTGAACAAG-TAMRA (SEQ ID NO: 73). Gene targetquantities were normalized using GAPDH expression levels. For humanGAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 74)reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 75) andthe PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 76)where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Primary human hepatocytes were treated with 150 nM of the compoundsshown in Table 4. Untreated cells served as the control to which alldata were normalized. Following 24 hours of treatment, apolipoprotein(a)expression levels were measured by real-time PCR as described herein,using the primers and probe described by SEQ ID NOs 71, 72 and 73. Thedata, shown in Table 4, represent the average of three experiments andare normalized to untreated control cells.

TABLE 4 Antisense inhibition of human apolipoprotein(a) using chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap:primary human hepatocytes TARGET ISIS SEQ TARGET % SEQ # REGION ID NOSITE INHIB ID NO 144367 Coding 4 174 77 11 144368 Coding 4 352 59 12144369 Coding 4 522 69 13 144370 Coding 4 1743 75 14 144371 Coding 42768 57 15 144372 Coding 4 2910 54 16 144373 Coding 4 3371 49 17 144374Coding 4 4972 80 18 144375 Coding 4 5080 11 19 144376 Coding 4 5315 8220 144377 Coding 4 5825 72 21 144378 Coding 4 6447 72 22 144379 Coding 47155 46 23 144380 Coding 4 7185 78 24 144381 Coding 4 8463 64 25 144382Coding 4 8915 58 26 144383 Coding 4 9066 79 27 144384 Coding 4 10787 028 144385 Coding 4 11238 94 29 144386 Coding 4 11261 61 30 144387 Coding4 11461 60 31 144388 Coding 4 11823 57 32 144389 Coding 4 11894 39 33144390 Coding 4 11957 0 34 144391 Coding 4 12255 57 35 144392 Coding 412461 50 36 144393 Coding 4 12699 82 37 144394 Coding 4 13354 76 38144395 3′UTR 4 13711 84 39 144396 3′UTR 4 13731 72 40 144397 3′UTR 413780 64 41 144398 3′UTR 4 13801 33 42 144399 3′UTR 4 13841 44 43 1444003′UTR 4 13861 75 44 144401 3′UTR 4 13881 72 45

Example 21 Effects of Antisense Oligonucleotides Targeted to HumanApolipoprotein(a) on Human Plasminogen Expression

Human apolipoprotein(a) sequence shares a high degree of homology withthe human plasminogen sequence. Thus it was of interest to determine ifantisense oligonucleotides targeting apolipoprotein(a) would exhibit aninhibitory effect on human plasminogen.

In a further embodiment, compounds designed to target humanapolipoprotein(a), shown in Table 1, were tested for their effects onhuman plasminogen mRNA expression. Pre-plated primary human hepatocyteswere purchased from InVitro Technologies (Baltimore, Md.). Cells werecultured in high-glucose DMEM (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum, 100 units per mLpenicillin, and 100 μg/mL streptomycin (all supplements from InvitrogenLife Technologies, Carlsbad, Calif.). Immediately upon receipt from thevendor, cells were transfected with a dose of 150 nM of antisenseoligonucleotide as described in other examples herein.

Following 24 hours of exposure to antisense oligonucleotides, humanplasminogen mRNA levels were measured by quantitative real-time PCR asdescribed in other examples herein. Probes and primers to humanplasminogen were designed to hybridize to a human plasminogen sequence,using published sequence information (GENBANK® accession number NM000301.1, incorporated herein as SEQ ID NO: 77). For human plasminogen,the PCR primers were:

forward primer: CGCTGGGAACTTTGTGACATC (SEQ ID NO: 78)reverse primer: CCCGCTGCACAACACCTCCACC (SEQ ID NO: 79)and the PCR probe was: 5′ JOE-CACTGGTAGGTGGGACCAGAA-TAMRA 3′ (SEQ ID NO:80) where JOE is the fluorescent reporter dye and TAMRA is the quencherdye. Gene target quantities were normalized using GAPDH expressionlevels.

Data, shown in Table 5, are averages from three experiments in whichprimary human hepatocytes were treated with antisense oligonucleotidestargeted to human apolipoprotein(a).

TABLE 5 Effects of chimeric phosphorothioate oligonucleotides targetedto human apolipoprotein (a) on human plamsinogen expression ISIS # %INHIB SEQ ID NO 144367 62 11 144368 49 12 144369 8 13 144370 44 14144371 0 15 144372 11 16 144373 33 17 144374 60 18 144375 9 19 144376 3220 144377 43 21 144378 8 22 144379 0 23 144380 31 24 144381 13 25 14438245 26 144383 47 27 144384 0 28 144385 0 29 144386 0 30 144387 0 31144388 36 32 144389 0 33 144390 0 34 144391 0 35 144392 0 36 144393 5837 144394 24 38 144395 35 39 144396 62 40 144397 25 41 144398 0 42144399 0 43 144400 60 44 144401 0 45

These data illustrate that ISIS 144371, 144379, 144384, 144385, 144386,144387, 144389, 144390, 144391, 144392, 144398, 144399 and 144401 do notinhibit plasminogen expression. Thus, in this assay, these compoundsselectively inhibit apolipoprotein(a) expression. ISIS 144369, 144378and 144375 demonstrated less than 10% inhibition of plasminogen. Thetarget sites in human apolipoprotein(a) to which ISIS 144379, ISIS144368 and ISIS 144376 bind share 70%, 70% and 80% nucleotide identitywith human plasminogen, respectively.

Example 22 Antisense Inhibition of Human Apolipoprotein(a) In Vivo:Transgenic Mouse Study

Apolipoprotein(a) is found in humans, nonhuman primates and the Europeanhedgehog, but not in common laboratory animals such as rats and mice.Accordingly, mice harboring a human apolipoprotein(a) transgene arerequired to investigate the effects of antisense oligonucleotides onhuman apolipoprotein(a) expression.

In a further embodiment, antisense oligonucleotides targeted to humanapolipoprotein(a) were tested for their effects in mice transgenic forboth human apolipoprotein(a) and human apolipoprotein B, as well as inmice transgenic for human apolipoprotein B alone. The transgenic micewere provided by Dr. Robert Pitas and Dr. Matthias Schneider in theGladstone Institute at the University of California, San Francisco.

Mice were treated with 25 mg/kg of ISIS 144379 (SEQ ID NO: 23), twiceweekly, for a period of 4 weeks. A control group consisting of micetransgenic for both human genes was treated with saline. Each treatmentgroup consisted of 4 animals. At the end of the 4 week treatment period,animals were sacrificed, and apolipoprotein(a) mRNA levels in livertissue were measured by real-time PCR, as described herein.Apolipoprotein B mRNA was also measured by real-time PCR with probes andprimers designed using published sequence information (GENBANK®accession number NM_(—)000384.1, incorporated herein as SEQ ID NO: 81).For human apolipoprotein B the PCR primers were:

forward primer: TGCTAAAGGCACATATGGCCT (SEQ ID NO: 82)reverse primer: CTCAGGTTGGACTCTCCATTGAG (SEQ ID NO: 83)and the PCR probe was: FAM-CTTGTCAGAGGGATCCTAACACTGGCCG-TAMRA (SEQ IDNO: 84) where FAM is the fluorescent reporter dye and TAMRA is thequencher dye. Gene target quantities were normalized using mouse GAPDHexpression levels, as described herein.

The data, shown in Table 6, represent the average of all animals in eachtreatment group and are normalized to saline-treated control animals.

TABLE 6 Antisense inhibition of human apolipoprotein(a) in transgenicmice mRNA expression % control Transgene apoB apo(a) apolipoprotein B101 0 apolipoprotein B 133 61 apolipoprotein(a)

These data illustrate that treatment of mice transgenic for humanapolipoprotein(a) and human apolipoprotein B with ISIS 144379 resultedin a decrease in apolipoprotein(a), but not apolipoprotein B, mRNAexpression.

Example 23 Antisense Oligonucleotides Targeted to Apolipoprotein(a)Having 2′-MOE Wings and Deoxy Gaps

In a further embodiment, and additional series of oligonucleotides wasdesigned to target the human apolipoprotein(a) sequence, using publicsequence information (GENBANK® accession #NM_(—)005577.1, incorporatedherein as SEQ ID NO: 4). The compounds are shown in Table 7. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. All compounds in Table 7are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines.

TABLE 7 Antisense oligonucleotides targeted to apolipo-protein(a) having 2′-MOE wings and a deoxy gap TARGET SEQ SEQ ID TARGETID ISIS # REGION NO SITE SEQUENCE NO 359474 5′ UTR 4 11cagtgtccagaaagtgtgtc 85 359475 Coding 4 12380 ggtttgctcagttggtgctg 86359476 Coding 4 12409 ttaccatggtagcactgccg 87 359477 Coding 4 12419actctggccattaccatggt 88 359478 Coding 4 12449 tgtgacagtggtggagaatg 89359479 Coding 4 12669 tgacagtcggaggagcgacc 90 359480 Coding 4 12839tgcccatttatttgtccctg 91 359481 Coding 4 12919 agttttcttggattcattgt 92359482 Coding 4 12944 gagagggatatcacagtagt 93 359483 Coding 4 13359cagtcctggcggtgaccatg 94 359484 Coding 4 13466 cttatagtgattgcacactt 95359485 Coding 4 13493 tctggccaaatgctcagcac 96

Example 24 Antisense Inhibition of Apolipoprotein(a) in Human PrimaryHepatocytes: Dose Response

In a further embodiment, antisense oligonucleotides targeted to humanapolipoprotein(a) were selected for dose response studies. Human primaryhepatocytes were treated with 25, 50, 150 and 300 nM of ISIS 144367,ISIS 144370, ISIS 144385, ISIS 144393 and ISIS 144395. ISIS 133529 wasused as a control oligonucleotide. Untreated cells served as the controlto which data were normalized. Following 24 hours of exposure toantisense oligonucleotides, target mRNA expression levels were measuredby real-time PCR as described by other examples herein. The results,shown in Table 8, are the average of 3 experiments and are expressed aspercent inhibition of apolipoprotein(a) expression relative to untreatedcontrol cells. “N.D.” indicates not determined.

TABLE 8 Antisense inhibition of apolipoprotein(a) in human primaryhepatocytes: dose response % Inhibition relative to untreated controlcells Dose of oligonucleotide ISIS # 25 50 150 300 144367 57 76 88 87144370 47 62 56 26 144385 33 36 59 39 144393 23 32 35 30 144395 34 35 3535 113529 N.D. N.D. 8 21

These data demonstrate that ISIS 144367 inhibited apolipoprotein(a) in adose-dependent manner. The other oligonucleotides tested were able toreduce apolipoprotein(a) expression.

Example 25 Effects of Antisense Inhibition of Apolipoprotein(a) onPlasminogen Expression: Dose Response in Primary Human Hepatocytes

In a further embodiment, antisense oligonucleotides targeted to humanapolipoprotein(a) were tested for their ability to inhibit humanplasminogen expression. Human primary hepatocytes were treated with 25,50, 150 and 300 nM of ISIS 144367, ISIS 144370, ISIS 144385, ISIS 144393and ISIS 144395. ISIS 113529 was used as a control oligonucleotide.Untreated cells served as the control to which data were normalized.Following 24 hours of exposure to antisense oligonucleotides, targetmRNA expression levels were measured by real-time PCR as described byother examples herein. The results, shown in Table 9, are the average of3 experiments and are expressed as percent inhibition ofapolipoprotein(a) expression relative to untreated control cells. “N.D.”indicates not determined.

TABLE 9 Effects of antisense inhibition, of apolipoprotein(a) onplasminogen expression in human primary hepatocytes: dose response %plasminogen expression relative to untreated control cells Dose ofoligonucleotide (nM) ISIS # 25 50 150 300 144367 0 0 0 0 144370 0 6 9 0144385 10 5 12 0 144393 10 39 2 0 144395 0 0 0 0 113529 N.D. N.D. 76 89

These data demonstrate that ISIS 144367 and ISIS 144395 did not inhibitthe expression of plasminogen in this assay and are thereforeapolipoprotein(a)-specific antisense oligonucleotides. ISIS 144370 andISIS 144385 did not result in a considerable reduction in plasminogenexpression.

Example 26 Effects of Antisense Inhibition of Apolipoprotein(a) inCytokine-Induced Cells

Elevated plasma levels of Lp(a), caused by increased expression ofapolipoprotein(a), is an independent risk factor for a variety ofcardiovascular disorders, including atherosclerosis,hypercholesterolemia, myocardial infarction and thrombosis (Seed et al.,N. Engl. J. Med., 1990, 322, 1494-1499; Sandkamp et al., Clin. Chem.,1990, 36, 20-23; Nowak-Gottl et al., Pediatrics, 1997, 99, E11).Furthermore, increases in plasma Lp(a) are associated with elevations inseveral acute-phase proteins, which participate in the acute-phase ofthe immune response and function to promote inflammation, activate thecomplement cascade, and stimulate chemotaxis of phagocytes. Thus, Lp(a)is proposed to be an acute-phase reactant and, consequently, responsiveto cytokines. The apolipoprotein(a) promoter contains several functionalcis-acting elements that are responsive to interleukin-6 (Wade et al.,Proc. natl. Acad. Sci. USA, 1993, 90, 1369-1373), a major mediator ofthe acute phase response, further suggesting a link between Lp(a) andthe acute phase response. An association between cytokines and Lp(a) wasobserved in primary monkey hepatocytes, where stimulation of the cellswith interleukin-6 resulted in an increase in Lp(a) protein, as well asin apolipoprotein(a) mRNA (Ramharack et al., Arterioscler. Thromb. Vasc.Biol., 1998, 18, 984-990). To date, no direct association betweencytokines and apolipoprotein(a) expression has been demonstrated inhumans. Thus, it is of interest to determine whether the antisenseinhibition of apolipoprotein(a) is affected by cytokine induction.

In a further embodiment, the ability of ISIS 144367 (SEQ ID NO: 11) toinhibit apolipoprotein(a) expression was investigated in primary humanhepatocytes which were induced with cytokines. For a period of 24 hours,cells were induced using culture media supplemented with a finalconcentration of 1 μM dexamethasone, 400 U/ml interleukin-1B and 200U/ml interleukin-6. At the end of this induction period, cells weretreated with oligonucleotide as described herein, for a period of 48hours. One group of cells was cytokine-induced and treated with 12.5,25, 50, 100 or 200 nM of ISIS 144367; data from these cells wasnormalized to data from cells receiving only cytokine treatment. Asecond group of cells received no cytokine induction and were treatedwith 12.5, 25, 50, 100 and 200 nM of ISIS 144367; data from these cellswas normalized to cells that received neither cytokine noroligonucleotide treatment. After the 48 oligonucleotide treatmentperiod, cells were harvested and apolipoprotein(a) expression wasmeasured by real-time PCR as described herein. The data, presented inTable 10, are the average of 3 experiments and are normalized to therespective controls as described. Results are shown as percentinhibition of apolipoprotein(a) expression.

TABLE 10 Antisense inhibition of apolipoprotein(a) in cytokine-inducedprimary human hepatocytes % Inhibition relative to control Dose of NoCytokine oligonucleotide (nM) induction induction 12.5 37 42 25 37 37 5042 62 100 75 87 200 65 89

These data demonstrate a dose-dependent reduction in apolipoprotein(a)expression cytokine-induced cells following treatment with ISIS 144367.In cells receiving no oligonucleotide treatment, the expression ofapolipoprotein(a) was similar in cytokine-induced cells relative tocells that were not exposed to cytokines. Furthermore, ISIS 144367inhibited apolipoprotein(a) expression to a greater extent incytokine-induced cells relative to cells not exposed to cytokines. Thus,ISIS 144367 is a more effective inhibitor of apolipoprotein(a)expression in cytokine-induced cells. These data demonstrate a linkbetween cytokine stimulation of primary human hepatocytes and theantisense inhibition of apolipoprotein(a) expression.

The expression of plasminogen was also tested in cytokine-induced cellsthat received ISIS 144367 treatment. Cells were induced and treated asdescribed for the apolipoprotein(a) mRNA expression experiment.Plasminogen mRNA was measured by real-time PCR as described herein. Thedata, averaged from 3 experiments and normalized to the appropriatecontrols, demonstrated that in this assay, in unstimulated cells as wellas cytokine-induced cells, ISIS 144367 did not inhibit plasminogen.Thus, the effects of ISIS 144367 are specific to apolipoprotein(a)expression both in the presence and absence of cytokines.

1. A compound comprising a modified oligonucleotide 12 to 30 linkednucleobases in length targeted to a nucleic acid molecule encodingapolipoprotein (a), wherein the compound comprises at least 8consecutive nucleobases of any of the nucleobase sequences of SEQ IDNOs: 85, 89-93, 95-96 and is at least 80% complementary to SEQ ID NO: 4.2. The compound of claim 1, wherein the modified oligonucleotide is atleast 85%, at least 90%, at least 95%, or 100% complementary to SEQ IDNO:
 1. 3. The compound of claim 1, wherein the modified oligonucleotideis single-stranded.
 4. The compound of claim 1, wherein at least oneinternucleoside linkage is a modified internucleoside linkage.
 5. Thecompound of claim 4, wherein each internucleoside linkage is aphosphorothioate internucleoside linkage.
 6. The compound of claim 1,wherein the modified oligonucleotide comprises at least one modifiedsugar.
 7. The compound of claim 6, wherein at least one modified sugaris a bicyclic sugar.
 8. The compound of claim 6, wherein at least onemodified sugar comprises a 2′-β-methoxyethyl, or a 4′-CH₂—O-2′.
 9. Thecompound of claim 1, wherein at least one nucleoside comprises amodified nucleobase.
 10. The compound of claim 9, wherein the modifiednucleobase is a 5-methylcytosine.
 11. The compound of claim 1, whereinthe modified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises: a gap segment consisting of linked deoxynucleosides; a 5′wing segment consisting of linked nucleosides; a 3′ wing segmentconsisting of linked nucleosides; wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment and wherein eachnucleoside of each wing segment comprises a modified sugar.
 12. Thecompound of claim 11, wherein the modified oligonucleotide consists of20 linked nucleosides and comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, wherein each internucleosidelinkage is a phosphorothioate linkage and wherein each cytosine residueis a 5-methylcytosine.
 13. The compound of claim 11, wherein themodified oligonucleotide consists of 20 linked nucleosides.
 14. Acomposition comprising the compound of claim 1, or a salt thereof, and apharmaceutically acceptable carrier or diluent.
 15. A compositioncomprising the compound of claim 1, for use in therapy.
 16. The compoundof claim 15, for use in treating, preventing, or slowing progression ofa disease related to elevated apo(a) and/or elevated Lp(a).
 17. Thecompound of claim 16, wherein the disease is an inflammatory,cardiovascular or metabolic disease, disorder or condition.